The overall goal of this research is to understand how adamantane antiviral drugs bind to the influenza A M2 protein. Influenza A virus is a major public health concern, both in its annual death toll and its potential to cause devastating pandemics. Although adamantane drugs have been in clinical use as antivirals for more than 40 years, there is still vigorous debate about how these drugs work. Circulating strains of influenza A are now largely resistant to the adamantane drugs. However, there is still intense interest is learning how these drugs bind to non-resistant strains of M2 protein and if structural information on the M2 protein can facilitate the rational design of the next generation of antiviral drugs. The majority of previously published biophysical work on M2 has focused on truncated M2 peptide constructs solubilixed in detergent or in bilayers of limited physiological relevance. To eliminate potential artifacts due to the use of fragments of M2 and distortion of the structure due to non-native hydrophobic environments, the work proposed here will use full-length protein reconstituted into membrane bilayers that reflect the lipid composition of influenza A virions. Our strategy for characterizing antiviral binding sites will rely on site-directed spin label (SDSL) EPR experiments designed to measure distances between bound-drug and spin-labeled sites throughout the protein. Using recently published high-resolution structural information on M2 fragments as a guide, we will prepare a series of single-site spin-labeled M2 proteins. Using deuterated forms of antiviral drugs, electron spin-echo envelope modulation (ESEEM) experiments will be used to measure distances between the deuterium nuclei on the drug and spin-labeled sites on the M2 protein. To complement the ESEEM distances, double electron-electron resonance (DEER) experiments will be used to measure distances between spin-labeled sites on the M2 protein and a spin-labeled drug. The high sensitivity of SDSL EPR studies will allow a range of drug:peptide:lipid ratios to be tested, enabling a fuller characterization of the location(s) and affinities of adamantane-binding site(s) than has been previously possible. Once binding site locations are determined, a battery of SDSL EPR data will be collected and used to determine drug-bound conformation(s) of the full length M2 protein in viral mimetic membranes. SDSL EPR data used in structure determination will include patterns of spin label mobilities, distance constraints from spin-spin couplings and accessibility to paramagnetic reagents of varying bilayer/aqueous solubility. PUBLIC HEALTH RELEVANCE: This proposal describes experiments designed to answer critical questions about how antiviral drugs bind to the influenza A M2 protein. Influenza A virus is a major public health concern, both in its annual death toll and its potential to cause devastating pandemics. Growing resistance to current antiviral drugs makes a full understanding of drug targets a priority.